Production and characterization of antibody against Opisthorchis viverrini via phage display and molecular simulation

In this study, a key issue to be addressed is the safe disposal of hybridoma instability. Hybridoma technology was used to produce anti–O. viverrini monoclonal antibody. Previous studies have shown that antibody production via antibody phage display can sustain the hybridoma technique. This paper presents the utility of antibody phage display technology for producing the phage displayed KKU505 Fab fragment and using experiments in concomitant with molecular simulation for characterization. The phage displayed KKU505 Fab fragment and characterization were successfully carried out. The KKU505 hybridoma cell line producing anti–O. viverrini antibody predicted to bind to myosin was used to synthesize cDNA so as to amplify the heavy chain and the light chain sequences. The KKU505 displayed phage was constructed and characterized by a molecular modeling in which the KKU505 Fab fragment and -O. viverrini myosin head were docked computationally and it is assumed that the Fab fragment was specific to -O. viverrini on the basis of mass spectrometry and Western blot. This complex interaction was confirmed by molecular simulation. Furthermore, the KKU505 displayed phage was validated using indirect enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry. It is worthy to note that ELISA and immunohistochemistry results confirmed that the Fab fragment was specific to the -O. viverrini antigen. Results indicated that the approach presented herein can generate anti–O. viverrini antibody via the phage display technology. This study integrates the use of phage display technology together with molecular simulation for further development of monoclonal antibody production. Furthermore, the presented work has profound implications for antibody production, particularly by solving the problem of hybridoma stability issues.

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Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology

Superantigens - Methods in Molecular Biology ◽

10.1007/978-1-4939-3344-0_17 ◽

2015 ◽

pp. 207-225 ◽

Cited By ~ 2

Author(s):

Pawan Kumar Singh ◽

Ranu Agrawal ◽

D. V. Kamboj ◽

Lokendra Singh

Keyword(s):

Phage Display ◽

Scfv Antibody ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Phage Display Technology ◽

Antibody Phage ◽

Antibody Phage Display ◽

Display Technology

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Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display Libraries

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108330113 ◽

2009 ◽

Vol 14 (3) ◽

pp. 282-293 ◽

Cited By ~ 38

Author(s):

Laura Turunen ◽

Kristiina Takkinen ◽

Hans Söderlund ◽

Timo Pulli

Keyword(s):

Phage Display ◽

High Efficiency ◽

Magnetic Bead ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Single Chain Antibody ◽

Phage Display Technology ◽

Antibody Phage ◽

Antibody Phage Display ◽

Display Technology

Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human γ-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 γ-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and β-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 β-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency. ( Journal of Biomolecular Screening 2009:282-293)

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generation of hapten-specific recombinant antibodies: antibody phage display technology: a review

Veterinární Medicína ◽

10.17221/5620-vetmed ◽

2012 ◽

Vol 50 (No. 6) ◽

pp. 231-252 ◽

Cited By ~ 23

Author(s):

J. Brichta ◽

M. Hnilova ◽

T. Viskovic

Keyword(s):

Phage Display ◽

Recombinant Dna ◽

Recombinant Antibodies ◽

Laboratory Animals ◽

Automatic Process ◽

Phage Display Technology ◽

Current Trends ◽

Antibody Phage ◽

Antibody Phage Display ◽

Display Technology

Production of antibodies has been revolutionized by the development of modern molecular biology methods for the expression of recombinant DNA. Phage display technology represents one of the most powerful tools for production and selection of recombinant antibodies and has been recognized as a valuable alternative way for the preparation of antibodies of a desired specificity. In comparison to poly- and monoclonal antibodies, recombinant antibodies using the phage display technology can be prepared faster, in more automatic process and with reduced consumption of laboratory animals. This review summarizes current trends of phage display technology with focus on the generation of hapten-specific recombinant antibodies and gives the examples of successful applications of phage display in the environmental analysis of low molecular weight compound.

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